The process of pre-mRNA splicing is carried out by a macromolecular complex, the spliceosome, the major components of which are the U1, U2, U5 and U4/U6 small nuclear ribonucleoprotein particles (snRNPs) (Kambach, et al., 1999, Curr Opin Struct Biol 9: 222-230; Will and Luhrmann, 2001, Curr Opin Cell Biol 13: 290-301; Patel and Steitz, 2003, Nat Rev Mol Cell Biol 4, 960-970; Nilsen, 2003, Bioessays 25: 1147-1149). Each of the U snRNPs (except for U6) is composed of one snRNA molecule, a set of seven common proteins and several proteins that are specific to individual snRNAs (Will and Luhrmann, 2001, Curr Opin Cell Biol 13: 290-301; Luhrmann, 1990, Mol Biol Rep 14: 183-192; Luhrmann, et al., 1990, Biochim Biophys Acta 1087: 265-292). SnRNP biogenesis begins with the transcription of the snRNAs in the nucleus followed by their nuclear export to the cytoplasm, where the major assembly process of the snRNP takes place. The common proteins, called Sm proteins, B/B′, D1, D2, D3, E, F, and G, are arranged into a stable heptameric ring, the Sm core, on a uridine-rich sequence motif (the Sm site) of the snRNAs (Branlant, et al., 1982, Embo J. 1: 1259-1265; Kambach, et al., 1999, Cell 96: 375-387; Stark, et al., 2001, Nature 409: 539-542; Achsel, et al., 2001, Proc Natl Acad Sci USA 98: 3685-3689). The assembly of Sm cores is required for the subsequent modification of the 7-methyl guanosine cap of snRNAs into a 2,2,7-trimethyl guanosine cap as well as for the stability and function of the snRNPs (Mattaj, 1986, Cell 46: 905-911; Plessel, et al., 1994, Mol Cell Biol. 14: 4160-4172). Properly assembled and modified snRNPs are then imported into the nucleus, where additional snRNP-specific proteins associate to form fully functional snRNPs (Will and Luhrmann, 2001, Curr Opin Cell Biol 13: 290-301; Mattaj, 1986, Cell 46: 905-911; Fischer and Luhrmann, 1990, Science 249; 786-790; Fischer, et al., 1993, Embo J. 12: 573-583; Hamm, et al., 1990, Cell 62: 569-577; Mattaj, et al., 1993, Mol Biol Rep 18: 79-83).
Earlier studies have demonstrated that snRNP assembly readily occurs in vitro with purified total snRNP proteins (TPs) and snRNAs (Sumpter, et al., 1992, Mol Biol Rep 16: 229-240; Raker, et al., 1996, Embo J. 15: 2256-2269; Raker, et al., 1999, Mol Cell Biol 19: 6554-6565) in an ATP-independent manner and without requirement for non-snRNP proteins. However, reconstitution of snRNPs in extracts from Xenopus laevis eggs and mammalian cells requires ATP (Kleinschmidt, et al., 1989, Nucleic Acids Res 17: 4817-4828; Temsamani, et al., 1991, J. Biol Chem 266: 20356-20362; Meister, et al., 2001, Nat Cell Biol 3, 945-949; Meister and Fischer, 2002, Embo J. 21: 5853-5863; Pellizzoni, et al., 2002, Science 298: 1775-1779), suggesting that snRNP assembly might be regulated by additional factors in vivo. Studies on a macromolecular complex containing the survival of motor neurons (SMN) protein indicated that the SMN complex is required for snRNP assembly (Temsamani, et al., 1991, J. Biol Chem 266: 20356-20362; Meister, et al., 2001, Nat Cell Biol 3, 945-949; Meister and Fischer, 2002, Embo J. 21: 5853-5863; Pellizzoni, et al., 2002, Science 298: 1775-1779; Fischer, et al., 1997, Cell 90: 1023-1029; Pellizzoni, et al., 1998, Cell 9: 615-624; Buhler, et al., 1999, Hum Mol Genet 8: 2351-2357; Yong, et al., 2004, Trends Cell Biol 14: 226-232). Experiments in cell extracts and with purified SMN complexes demonstrate that the SMN complex mediates the assembly of Sm cores (Meister, et al., 2001, Nat Cell Biol 3, 945-949; Meister and Fischer, 2002, Embo J. 21: 5853-5863; Pellizzoni, et al., 2002, Science 298: 1775-1779). SMN is the protein product of the gene responsible for spinal muscular atrophy (SMA), a common and often fatal genetic disorder in which motor neurons in the spinal cord degenerate (Lefebvre, et al., 1995, Cell 80: 155-165; Cifuentes-Diaz, et al., 2002, Semin Pediatr Neurol 9: 145-150; Iannaccone, et al., 2004, Curr Neurol Neurosci Rep 4: 74-80; Crawford, et al., 1996, Neurobiol Dis 3: 97-110). Based on the age of onset and the severity of the disease, SMA is clinically classified into three types: the severe type I, the moderate type II and the mild type III. The severity of SMA clinical phenotypes is closely linked to the degree of reduction of SMN protein levels in SMA patient-derived cell lines (Lefebvre, et al., 1997, Nat Genet 16: 265-269; Coovert, et al., 1997, Hum Mol Genet 6: 1205-1214).
Depletion experiments demonstrated that the SMN complex is required for snRNP assembly, but what happens in patients' cells, where the amount of the protein is reduced to varying degrees, has not been determined. Current methods of monitoring snRNP assembly are not suitable for quantitative analysis.
In addition, snRNP assembly is an excellent model for additional interactions between an RNA binding protein and RNA (RNA binding protein-RNA)U. Where the amount of the RNA binding protein is reduced to varying degrees, or activity is otherwise impaired or inhibited, pathogenesis ensues. However, current methods of monitoring RNA binding protein-RNA interaction and RNA binding protein assembly are not suitable for quantitative analysis.
There exists a long felt need to develop rapid, accurate and automatable assays to analyze snRNP assembly to determine the effect that SMA, as well as other diseases and conditions, have on the initiation of transcription. In addition, there exists a long felt need to develop rapid, accurate and automatable assays to analyze RNA binding protein-RNA interactions and the effect that the activation or inhibition of these interactions has on disease states. The present invention meets this need.